Method and apparatus for keeping artificially created tissues alive

ABSTRACT

A device to aid in the production and regeneration of tissues and organs, standing alone, or on or inside of the human body, with a method of fabrication of tissues and organs and use of the device.

CROSS REFERENCE TO RELATED APPLICATIONS

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FEDERALLY SPONSORED RESEARCH

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NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATE BY REFERENCE CD OF SEQUENCE LISTING OR PROGRAM LISTING

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PRIOR DISCLOSURES BY INVENTOR OR JOINT INVENTOR

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BACKGROUND 1. Field of the Invention

Generally, the invention relates to biomedical research and development,and biomedical engineering of living tissues including: laboratory ormedical devices that produce living artificial tissue, whether human ornot, such as hearts or kidneys, etc. More specifically, the inventionrelates to keeping these tissues alive for sustained periods of timeusing a scaffold and vascular structure consistent with the tissues'authentic three-dimensional architecture, so that these tissues may beused in applications such as laboratory testing, transplantation, etc.

2. Background Art

A tissue is an aggregate of cells that form a definitive type ofstructural material having a specific biological function. Although atissue can comprise a single cell type, a tissue typically comprisesmore than one cell type; this is referred to as a heterogeneous tissue.

The extracellular matrix is a pathway for molecular communication withinand between cells, between neighboring tissues, between organs, and withmigratory cells by way of: nerves, blood and lymphatic vessels, movementof small molecules in and between cells/tissues/organs, the directcontact of signaling molecules secreted by or presented on the surfaceof neighboring cells of the same tissues, from different organs or fromthe signal orchestrated movement of stem cells, or on fibers of theextracellular matrix itself, providing a mechanism for response tomechanical stimuli. Examples of tissue structures given mechanical,physiological, and functional support by an extracellular matrixinclude, but are not limited to: blood vessels, nerves, motor endplates, capillary beds, collecting tubules of the kidney, glomeruli,Bowman's capsule, and islets of Langerhans.

Additionally, the basement membrane system is a specialized connectivetissue structure found in all tissue systems. The basement membranesystem forms a continuous structure, unifying the vascular system—thecirculatory interface necessary to sustain three-dimensional tissuestructures. The basement membrane system is a platform essential fordesigning extra-cellular compartments used by the organs to housevarious cell types, which make up the organ's structure.

This chemical-engineering data influences how cells move, grow, anddifferentiate based on tissue type. These activities are responsible forchanges in cell form and structure that take place when new tissues areformed and when damaged tissues are restored to good health.

The loss of or failure of a tissue or organ requires repair orreplacement of the tissue. Tissue loss can result from, for example,injury (e.g., burns), disease (e.g., diabetes), and congenital defects.Damaged tissues/organs are often replaced with donor tissue/organs.This, however, is expensive and, moreover, there is a severe shortage ofdonor tissue/organs. Indeed, there is a long waiting list in thiscountry for persons needing an organ transplant. Tissue engineering is apotential solution to address this shortage.

Tissue engineering is a technique that has the potential to create new,living tissues and organs. Tissue engineering involves providing ascaffold that can be seeded with cells for a given type of tissue thatwill allow the cells to develop into a tissue. The scaffold provides amechanical support for the cells to grow on. During development of thetissue, the scaffold degrades or is metabolized, eventually leaving anengrafted tissue in its place. Current advances in scaffold fabricationtechniques have allowed the formation of three-dimensional scaffolds.

A limitation in tissue engineering is that scaffolds fabricated bycurrently available methods lack the structural and material specificityto allow complex, heterogeneous tissue structures to be assembled. Thus,although scaffolds for developing engrafted tissues are known in theart, the tissues engineered using these scaffolds typically lack thefull functional capacity of the natural tissue, due to the inability ofthe known methods to create the intricate vascular support system. Itremains a major challenge to create three-dimensional tissue structureswith precise external dimensions and internal architecture.

Initially, polymeric systems, developed with polymer scaffolds thatreleased angiogenic factors, were used to recruit precursor cells intoporous scaffolding, but these materials lacked a specific form, whichwould lead to thrombosis, chronic inflammation, or total rejection upontransplantation. Additionally, many of the current scaffold fabricationtechniques do not allow direct incorporation of tissue-specific growthfactors, without compromising bioactivity. As a result, artificiallycreated tissues die quickly; therefore, not being able to be used intransplants or sustained for research purposes such as the developmentand testing of pharmaceuticals.

Since the early 1990's, with the onset of digital imaging and imageprocessing software, such as Adobe Photoshop, the goal for manufacturersof Micro Electro Mechanical Systems (MEMS) was to create high-resolutiondigital photographs that were indistinguishable from traditionalphotography. Continued advancement in MEMS has led to the production ofa charge coupling device (CCD chip) which captures images electronicallyat resolutions that are now exceeding the resolution of photographicfilms. Today's mid-level cameras can easily produce and processhigh-resolution images, which are very manageable on modern hardwaresystems. The high production capacity and the relative ease ofmanipulating images captured digitally has led to the production ofimage capturing systems in the medical industry, which traditionally usefilm and chemical processing to develop images. With present day imagecapturing systems, a big limit to the number and detail of images thatcan be captured, is the electronic hardware's ability to store andprocess data.

The need for noninvasive viewing of human organ structures in threedimensions quickly steered developers of imaging devices to produceequipment and software for the digital capture and rendering of thesethree-dimensional images for medical purposes. The quality of theimaging devices drove the development of instrumentation that digitallycollects images of the human architecture at very high resolution, butthe amount of image information captured becomes very limited due to theenormous amount of data and the lack of computer hardware capable ofmanaging it in real time. These developments are apparent in areas suchas x-ray computed tomography with the introduction of micro-computertomography. Micro-computer tomography (micro-CT) is commonly used togenerate three-dimensional tissue structures, but has found limitedsuccess because of the inability to reconstruct complete and accuratecapillary beds.

Another main limitation to developing artificial tissues is theinability to mimic the microscopic architectures that is essential tothe support, growth, maintenance, and function of the tissue structures.The difficulty with bioengineering blood vessels causes many researchersto focus strictly on the engineering of vascular tissues. Researchershave been unable to recreate vascular systems in three dimensions; thusforcing them to create several two dimensional sheets of tissues andlayering them, in order to create a third dimension. Additionally, noone has successfully been able to manufacture blood vessels smaller thansix (6) millimeters. This leads to limited functionality of the tissueand lacks the vascular organization necessary to produce variations inother organs and tissue structures.

Finally, many injuries in humans involve extensive loss of soft tissueswhich cannot be regenerated or repaired. Many large wounds requiretraumatic grafts or total amputation. The human body does not have theability to regenerate itself due to its large body plan. If soft tissueregeneration could be enhanced, then the limb could be spared. While thelarge body plans of many vertebrate organisms have made it possible tomineralize tissue in order to support the soft tissue, the cellularunits have remained restricted in size. As the size of the bodyincreases, so does the area that cells have to traverse in order tocover large wounds and have become incapable of initiation of theformation of the blastemas needed for limb regeneration, as seen insmaller organisms.

What is needed is an apparatus and method to provide the intricatescaffold and vascular structure required to keep engineered tissuesalive. Your applicant's method and device address the need to acquireboth microscopic detail and volume, and establishes a digital archivefor use in the reverse engineering of three-dimensional organstructures.

SUMMARY

In view of the foregoing disadvantages inherent in the creating ofartificial tissues, your applicant has devised a device for keeping thetissues alive and a method for using the device. Hereinafter, saiddevice will be referred to as the vascular bed chamber. The method isdesigned to systematically orchestrate coordinated events at differentstages of a scaffold's production and subsequently the different eventsoccurring during heterogeneous tissue development.

The method applies bio-computer aided design of cellular architecture tothe use of computer aided design models as digital manufacturing codecarriers that your applicant has termed: Chroma Coding or Chroma Codedcode (CC-code). This CC-code is designed to carry data pertaining to thenecessary environmental conditions needed to produce transformativeresults in tissue engineering, as well as Cartesian coordinates used forguiding robotic three-dimensional printers and three-dimensionalfabricating lasers. In addition, the code will allow the user to producemodels that interface directly and independently with computer guideddevices to supply material such as stem cells and growth factors, andconditions such as flow rate and temperature, to the product during themanufacturing process. As a result, computer aided design models of thevascular supply systems will include complete and accurate replicationsof the capillary bed systems. These vascular systems will then supportorgan structures based on their functional morphology.

The tissues can be grown on the scaffold either in vitro (as in vascularbed chamber, configuration one, described below) to produce anengineered tissue, or in vivo (as in vascular bed chamber, configurationtwo, described below). Where other approaches to tissue engineering seekto design scaffolds with physical characteristics that support andmaintain cell growth, your applicant has devised an approach toreplicate, from material imaging data, the structural designs of thebasement membrane and its interconnective extracellular supportstructures that are naturally found supporting the cells in the tissuestructures sought to be engineered. This allows for the complete andaccurate replication of the vascular structure of a tissue or organ to adegree previously thought unachievable.

The basement membrane is generally composed of two layers, oneglycoprotein rich, and the other rich in collagen proteoglycans. Thisstructure originates from the embryonic mesenchyme and surrounds allstationary cells units. Through hemidesmosomes, integrins andglycoprotein laminins, the basement membrane interfaces the cellularelements that form the smallest structural subunits in the body'stissues and organs. Along with its associated connective tissueframework, the basement membrane embraces and supports cells into unitswhich are ultimately coupled by associated connective tissues intoorgans, forming multicellular organisms. It is this array ofextracellular matrix surrounding organs that forms the grid ofpositional information, controlling pattern formation for regeneration.

The method and device also allow for regeneration of tissues and organsdirectly on or inside of the human body, as a regenerative process. Theregenerative process for limbs or nervous tissue is promoted by creatingthree-dimensional stromal and cellular structures, along withappropriate embedded signaling or trophic molecules to mimic theblastema-associated limb regeneration process. Successful regenerationrequires replacement of both structure and information.

The goal of regeneration is restoration of function. The limb must haveregenerated muscles which are coordinately patterned relative to thejoints and long bones to allow for flexion and extension, and theregenerated nerves must be patterned so as to coordinately innervatemuscles. Therefore, successful regeneration requires manipulation ofsignaling to induce dedifferentiation to multipotent blastema cells toremake the structure, and to provide spatial information to restorefunction.

The novelty of this process is that it combines image data thatrepresents both macro- and microscopic domains of tissue structures. Itthen uses that data for the production of the bio-informative blueprint(or Bio-CAD) which is designed to successfully interface with cells tosupport, recruit, and influence cellular behavior, mimicking theirnaturally developed tissue structures. Additionally, these bio-CADmodels are converted into data files that interface with programs whichdrive three-dimensional fabrication and micro patterning equipment.Properly pursued, the reconstruction of tissue images intohigh-resolution three-dimensional CAD models that contain complete andaccurate three-dimensional details of organ structures on a microscopiclevel is completely obtainable. With this method, the user is able tocreate tissue scaffolds and molecular patterned structural pathwayswhich replicate the structural design, compelling and supporting thegenesis of vascular tissue structures.

The three-dimensional digital image of bodily tissues can be obtained bya variety of methods including, but not limited to: micro-, macro-, andnano-computer tomography, nuclear magnetic resonance imaging, confocalmicroscopy, or reconstruction of serial sections using microscopicimages. By using actual image data obtained directly from vascular treesystems, the user can create a highly detailed model of its structure.

Given the proper image data, computer aided design tools can be used tocreate models that accurately replicate the basement membrane system'sinterfacing of cellular boundaries in animal tissues. Tissuebio-fabrication begins with atomic three-dimensional images of vascularcorrosion casts, representations of the geometry of the originalvascular system. The images of the casts are converted into computeraided design models and combined with developmental chemical engineeringdata in a bio-informative blueprint, capable of guiding the computeraided manufacture of tissues, using three-dimensional roboticbio-printers and multi-photon lasers. The data in the bio-informativeblueprint instructs computer aided manufacturing tools to createenvironments in bioreactors that mimic molecular mechanisms and cellularinteractions that would normally take place in the human body whentissue is being developed or regenerated.

The bio-informative blueprint uses micro-prototyping techniques such as:the three-dimensional chemical patterning of photo-cross linkablemultilayered hydrogels, the complete structural fabrication of vascularscaffolding—scaffolding that will support, nurture and guide thecellularization of microvascular and macrovascular structures, and serveas a nurturing framework. The scaffold can be divided into a pluralityof regions, and each region can have a different structure. The termstructure, in this context, refers to the characteristics of the regionthat enable a specific tissue type to develop at that region. Thestructure at a region not only includes the components that provide astructural support for the tissue to grow at that region, but can alsoinclude other components that facilitate cell growth such as, but notlimited to: growth factors, nutrients, and patterned development ofvascular and lymphatic support systems and nerve fibers, with theircorresponding connections to the central nervous, cardiovascular, andlymphatic systems. Additionally, this framework supports the furtherengineering and design of organ-specific, functional tissue structuresfrom chemically and mechanically engineered extracellular matrices,using micro-rapid prototyping and three-dimensional laser pulsepatterning for the regulation of tissue specific cellular morphologies.

The resulting scaffold provides a support that allows the growth of atissue. The scaffold provides an environment that guides growth of eachindividual tissue type, so that each tissue type is geometricallyarranged to recreate the tissue's unique cellular design and toreestablish the complex biological interrelationships that areresponsible for a given tissue's function. The method permits differentregions of the scaffold to be strategically composed of specificmaterial(s) that have structure and components that are conducive to thegrowth of a particular type of cell. The scaffold obtained by yourapplicant's method can, for example, include regions that are conduciveto growing blood vessels. Scaffolds obtained by typical fabricationprocesses cannot provide for the growth of blood vessels.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be best understood by those having ordinary skill inthe art by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings in which:

FIG. 1 illustrates a sectional view of the first embodiment of theapparatus in an expanded position for supporting the creation of tissuesor organs outside of the body.

FIG. 2 illustrates a perspective view of the first embodiment of theapparatus in an expanded position for supporting the creation of tissuesor organs outside of the body.

FIG. 3 illustrates a perspective view of the first embodiment of theapparatus in an expanded position, without the cover, to aid inexpansion for supporting the creation of tissues or organs outside thebody.

FIG. 4 illustrates a close-up, perspective view of the support bar thataids in the expansion of the first embodiment for supporting thecreation of tissues or organs outside of the body.

FIG. 5 illustrates a perspective view of the first embodiment within asupport chamber to show the interrelationship between the firstembodiment in an upside down collapsed state and the support bar thatexpands the first embodiment for supporting the creation of tissues ororgans outside of the body.

FIG. 6 illustrates a sectional view of the first embodiment with acompleted artificial heart.

FIG. 7 illustrates a perspective view of the second embodiment placeddirectly on an open wound for the direct repair of tissues or organs ona human body.

FIG. 8 illustrates a perspective view of the blastema-inducing filmcreated to overlay the wound and stimulate tissue or organ growthdirectly on a human body.

FIG. 9 illustrates a mold of the wound to be repaired containing aheating element to mold the film from FIG. 8 to be a direct overlaymatch of the actual wound for tissue or organ repair directly on thehuman body.

FIG. 10 illustrates the interlocking sections that make up the secondembodiment with inflow channels and outflow ports shown, for tissue ororgan repair directly on the human body.

FIG. 11 illustrates a sectional view of an embodiment for the repair ofdigits to be attached to the second embodiment for repair of tissues ororgans directly on the human body.

FIG. 12 illustrates a bottom view of a section comprising the secondembodiment with inflow ports and channels shown, as well as the outflowport shown at the top of the figure, for tissue or organ repair directlyon the human body.

FIG. 13 illustrates a bottom view of several sections that comprise thesecond embodiment with inflow channels and ports shown, as well as theoutflow ports, for tissue or organ repair directly on the human body.

FIG. 14 illustrates the input port with tubules extending from thebottom of the port directly on an open wound that will allow for theinflow of cells and other substrates for tissue and organ repairdirectly on the human body

FIG. 15 illustrates the inlet port that is expandable, collapsing overthe tubules which are foldable, to allow extraction from the wound fortissue or organ repair directly on the human body.

FIG. 16 illustrates the laser fabrication goggle, with a transparentopening at the bottom, which houses the microscope objective lens duringthe repair of tissue or organs directly on the human body.

FIG. 17 illustrates the microscope objective lens in the goggle withimmersion oil to aid in repair of tissue or organs directly on the humanbody.

FIG. 18 illustrates the completed form of the laser fabrication goggle,including the microscope objective lens, that will aid in repair oftissue or organs directly on the human body.

FIG. 19 illustrates a flow chart of the method from the beginning stageof which tissue to regenerate, through the computer modeling and tissuepreparation, all the way to the finished product either on/in thepatient's body, or ready for transplantation.

DRAWINGS—REFERENCE NUMERALS

-   10 Vascular Bed Chamber first configuration (VBC-I)-   12 Top of VBC-I-   14-20 Tapered expandable/collapsible sides-   22 Base-   24 Input/output tube

24 a Input port

24 b Output port

-   26 Input/output tube

26 a Output port

26 b Fabrication site

-   28 Input tube

28 a Outflow into chamber

28 b Input port

-   38 Locking spacers-   100 Production/Inversion chamber for housing VBC-I-   102 Support rod-   104 Sleeve to support slider (112)-   106 Adjustable slider-   108 Upper clamps attached to the support rod (102) to hold the    bottom of the inverted VBC-I during production-   110 Spring loaded lower clamps to hold the top of the inverted VBC-I    during production-   112 Slide that allows support rod to move in a vertical plane-   114 Inflow port to attach to inflow port 24 directly on VBC-I-   116 Outflow port to attach to outflow port 28 directly on VBC-I-   120 Adjustable knob attached to support rod to allow for the    vertical movement of support rod-   122 Thermal regulated stage at the base of inversion chamber to    support VBC-I-   124 Outflow port to attach to outflow port (24) directly on VBC-I-   126 Outflow port to attach to outflow port (26) directly on VBC-I-   128 Additional, optional port if needed-   130 Transparent portion in middle of base-   132 Seal between the thermal regulated base (122) and the    transparent middle section (130)-   200 Vascular Bed Chamber second configuration (VBC-2)-   202 Patient wound-   204 Blastema Induction Film-   206 Individual sections that fit together to form the entirety of    VBC-2-   208 Inner support tube for flow of cells and substrates into small    tubules for production-   210 Fabrication laser-   212 Inward flow tube for cells and other substrates-   214 Lip allowing (212) to slide over (208)-   216 Threads for attaching additional sections or digit sections    (244)-   218 Outflow ports for recirculation-   220 Protective hydrophilic gauze-   222 Fatty acid mixture with intergins-   224 Wax paper-   226 Division creating hydrophobic and hydrophilic regions of    intergins-   228 Hydrophilic region-   230 Hydrophobic region-   232 Wound mold-   234 Heating element-   236 Circulation channels-   238 Female opening for section attachment-   240 Male protrusion for section attachment, locks in to (238)-   242 Inflow ports-   244 Chamber for regeneration of digits for attachment to VBC-2    sections (206) via ports (216)-   246 Retractable tubules for the placement of cells and other    substrates directly on an open wound-   248 Outflow ports on tubules (246)-   250 Female threads on laser fabrication goggle-   252 Transparent base on laser fabrication goggle-   254 Laser fabrication goggle with immersion oil for encasing    microscope objective lens

DETAILED DESCRIPTION

Many different systems having features of the present invention arepossible. The following description describes the preferred embodimentof select features of those systems and various combinations thereof.These features may be deployed in various combinations to arrive atvarious desired working configurations of systems.

Reference is hereafter made to the drawings where like referencenumerals refer to like parts throughout the various views.

Method

The best description of the method is blastema based regeneration, butthe procedure is not that simple. It could also be considered computeraided tissue engineering/biofabrication, but not in the traditionalsense. This method enables the digital manufacturing of compositestructures, optimizing automated use of the simplest design up to thelargest data sets, allowing for production schemes on any scaleimaginable. The following things will be needed to produce blastemalbased regeneration of tissues: three-dimensional reconstructions ofsectioned tissues and castings of the tissues, computer aided design ofcellular architecture, three-dimensional regenerative blueprint, roboticproduction of a bio-stimulatory scaffold, vascular bed chamber (eitherfree standing—VBC-I, or form fitted to the patient's specificneeds—VBC-2).

Tissue bio-fabrication begins with atomic three-dimensional images ofvascular corrosion casts, representations of the geometry of theoriginal vascular system. The images of the casts are then convertedinto computer-aided design models and combined with developmentalchemical engineering data in a bio-informative blueprint, capable ofguiding the computer-aided manufacture of tissues usingthree-dimensional robotic printers and multi-photon lasers. With thisprocess, the production of these composite structures can bebio-manufactured digitally, producing cellular-based advanced materialsthat can regulate the regeneration of three-dimensional tissuestructures or organs.

The preferred method for obtaining the three-dimensional image isdecellularization of tissues, leaving the extracellular matrix. Thenhaving a clear picture of the matrix by staining the matrix with heavymetals, such as osmium, and capturing the images of the extracellularmatrix at three levels of resolution, using conventional computertomography, micro- and nano-CT, and reconstructing the tissue'sextracellular matrix from serial scans using a progressive removal oflayers of the extracellular matrix.

Given the proper image data, computer aided design (CAD) tools can beused to create models that accurately replicate the basement membranesystem's interfacing cellular boundaries which create the natural,modular compartmentalization found in animal tissues. Where otherapproaches to tissue engineering seek to design scaffolds with physicalcharacteristics that support and maintain cell growth, your applicant'smethod is to replicate, from medical imaging data, the structuraldesigns of the basement membrane and its inter connective cellularsupport structures (also known as the septa) that are naturally foundsupporting the cells in the tissue structures sought to be engineered.

These modeling results can be the basis for designing a bio-blueprint.The bio-blueprint uses micro-prototyping techniques, including but notlimited to: the three-dimensional chemical patterning of photo-crosslinkable multilayered hydrogels. This allows the bio-blueprint to guidethe more complete structural fabrication of a vascularscaffolding—scaffolding that will support, nurture and guide thecellularization of microvascular and macrovascular structures and serveas a nurturing framework. This framework supports the furtherengineering and design of organ-specific, functional tissue structuresfrom chemically and mechanically engineered extracellular matrices,using micro-rapid prototyping and three-dimensional laser pulsepatterning for the regulation of tissue specific cellular morphologies.These steps create a virtual design of the tissue, or a bio-CAD, andonce the bio-CAD has been completed it can be archived in a virtuallibrary and establish a virtual tissue bank.

Using CAD, these archived models can then be manipulated, with minimaleffort, to meet a patient's specific needs. A very high level ofprecision, capable of working at microscopic dimensions, facilitates themanufacture of tissue structures and/or extracellular matrices that willsupport bio-fabrication. This procedure leads to the production ofauthentic tissue structures for use in partial or total organ or tissuereplacement or transplant therapy.

The method can be broken down into the following steps, which will beelaborated on, individually, below.

-   -   1. Select organ/tissue structures.    -   2. Isolate the organ or tissue's vascular structures using        vascular casting.    -   3. Isolate the organ or tissue's septa systems, including the        organ or tissue's authentic encapsulating boundary structure        (for example: pericardium, endocardium, periosteum, endosteum,        perineurium, endoneurium, etc), by de-cellularization and or the        selective staining of its septa systems    -   4. Capture image of the septa, compartmenting connective tissue        boundaries, and vascular systems    -   5. Use images above to create three-dimensional models of the        structure    -   6. Import the three-dimensional models into computer-aided        design software    -   7. Repair the models using CAD tools and convert mesh into        non-uniform rational B-spline models    -   8. Designate the boundary where the septa and vascular cast        interfaced cells as site of the basement membrane    -   9. Use computer-aid design (CAD) tools to clean up the basement        membrane system's boundaries and model the walls of the vascular        tree system for the scripted production schema    -   10. The tissue's structures are broken down into their        structural sub-layers/subunits. For each sub-layer/subunit        categorize:        -   the cell types that form the layers/subunits,        -   the structural and functional material constituting in the            extracellular matrix within each type of layer/subunit,        -   growth factors needed to induce cells to produce the            different cell and extracellular matrix types that form            tissue structures within the layers/subunits        -   cytokines/chemotactic factors that need to be released to            attract native adult stem cells from the body's tissues into            specific sites of the fabricated scaffold        -   extracellular signaling factors such as structural molecules            with ligands that signal the cells to form tissue structures            within the layers/subunits            -   the functional peptides that constitute the above                ligands responsible for the extracellular signaling                factors                -   determine which ones need to be attached to the                    scaffold                -   patterning their attachment location within the                    scaffold            -   Cells used in the natural developmental/regenerative                processes            -   Cells to be used in fabrication processes            -   Encapsulating extracellular signaling factors within                molecular cages/nanospheres            -   Laser and biochemical techniques for attaching molecular                cages/nanospheres to the scaffold during and after the                bio-printing processes            -   Laser, bio-degenerative and enzymatic techniques for a                patterned time release of functional peptides, growth                factors and chemotactic factors from molecular                cages/nanospheres at scripted time intervals            -   Biochemical and laser techniques for attaching                functional peptides to scaffold    -   11. Chart the epigenetic information on the organ's/tissue        structure's natural development/regeneration to correspond with        the available CAM tools, their specifications, construction        materials and supported bioreactor processes    -   12. Use the chart from step 11 to script into Bio-CAD code for a        production plan producing and transmitting signals that control        the computer-aided manufacturing/tissue engineering tools'        robotic placement of construction materials; robotic positions        and triggering of laser tools for molecular/atomic manipulation        of said materials; bio-support of living fabricated structures        using vascular and interstitial fluid circulation;    -   13. Time the stimulation of tissue growth by regulating        environmental factors, forces applied to, temperature,        concentration chemicals of developing structure using fluid        circulation and laser motivated chemical reactions to coordinate        with feedback sensors for temperature, pressure and chemical        concentration of oxygen and carbon dioxide.    -   14. Map the expression of growth/differentiation factors    -   15. Use the Bio-CAD blueprint to coordinate the        delivery/triggered release of conditioned growth media, cells,        chemotactic and developmental growth factors with the        robotically controlled manufacturing process to mimic and induce        the cellular environments that naturally occur in the        developmental/regenerative tissue structure.    -   16. Modify Bio-CAD design for using CAD tools to create paths        and locations for each fabrication step. Duplicate CAD structure        for each independent fabrication step        -   a. Replicate the vascular tree model; make it a solid form        -   b. Offset model's location in the septa to model the            paralleling nerve fibers found in the tissue's septa.        -   c. Again replicate the vascular tree model and offset its            location in the septa to model the paralleling lymphatic            vessels, alternatively lymphatic systems can be cast and            modeled uniquely.            -   i. Tubular systems i.e. kidney collecting tubules, loops                of Henley, where present, are cast and modeled                separately.            -   ii. Size and position the locations of each of these                models to locations in the modeled septa to correspond                with their natural size and location.    -   17. Use Bio-CAD and apply Cartesian coordinates to each sub        model as creating unique groups of coordinates for conveying        scripted roles to CAM instrumentation in the production schema.    -   18. Associate the production schema with the appropriate        Cartesian coordinates to programs that guide CATE Strategies and        CAM Tools by:        -   a. Coding the production scheme for each uniquely modeled            tissue structure by creating a table with 18 single digit            values of 0-9.        -   b. Assign the code to the materials and the processes used            in the production scheme for fabricating each structure in            the CAD model.        -   c. Divide table into six categories each represented by            three digits.        -   d. Assign categories to each of the following color            components: Hue; Saturation; Value; Red; Green; Blue.        -   e. Use a common graphic software color picking tool to            covert the above code into color.        -   f. Apply the computed color above to the boundary            representation for each set of Cartesian coordinates            established to represent a tissue's structures.    -   19. CAM tools take instructions from the colors used in CAD        blueprints by decoding the color values at specific Cartesian        coordinates assigned to a tool.

The first step requires identifying the composite tissue and collectingthe image data on the structure. Organs will be injected with materialthat shows up on x-ray, either within the intact organ so thatsubsequent histological analysis can be performed and relatedanatomically to the vessels, or with a casting material that can beisolated from the tissue by selective dissolution of the tissue with acaustic solution. This casting method allows for higher spatialresolution. For organs where different vascular or tubular structureshave to be differentiated (for example: bronchioles from blood vessels,bile duct from hepatic vessels, or the portal vein from the hepaticartery, etc), contrast/casting materials doped with nano-particlesconsisting of different metals should be used so that the different Kabsorption edges can be identified. Additionally, the sample should bescanned in the X and Y planes, and different layers will be fabricatedby scanning the sample in the Z direction; thus, allowing nanostructuresto be fabricated. Three-dimensional laser patterning enables models,created following this process, to create a structural framework thatboth physically and biochemically guides the genesis of the originalorgan structure by incorporating factors responsible for stimulatingoptimal cellular and sub-cellular responses that mediate cell behaviorfor bioengineering the tissue structure.

Once the tissue or organ to be replaced has been identified, a vascularscaffolding will be designed directly from three-dimensional image data.This complete vascular network will also include the capillary systemsnecessary to feed the surrounding tissues. Sections are then convertedto three-dimensional wire frame models and are then merged togetherusing computer aided design (CAD) software. Reconstructed image datasets from Micro-CT and serial sectioning of the corresponding capillarybeds are combined to complete the framework for the CAD model (alsoreferred to as a bio-CAD model, or a bio-informative blueprint, orbio-blueprint). This produces a framework in a completethree-dimensional layout of the inner wall of the vascular tree systemin the tissue or organ of interest. CAD can then be used to render, ontop of the layout for the lumen wall, a design for structuralscaffolding structured for seeding migrating progenitor and/or stemcells. The data in the bio-CAD will instruct computer aidedmanufacturing tools to create environments in bioreactors, that mimicmolecular mechanisms and cellular interactions that would normally takeplace in the human body when tissue is being developed or regenerated.Then CAM uses three-dimensional robotic bio-printers and multi-photonlasers to produce an authentic three-dimensional tissue structure.

A bio-CAD blueprint is formed using a system comprising at least onecomputer that uses data collected on the authentic microscopicarchitecture of tissue structures from tissue specimen preparationtechniques that include but are not limited to:

-   -   Lumenal corrosion casting used to create luminal boundaries such        as: vascular lumen, endocardium, gastric lumen, intestinal        lumen, etc; and    -   Decellularization and molecular labeling of marginal boundaries        in cell structures such as: connective tissue capsule,        connective tissue septa, myocardium, pericardium, endocardium,        etc.

Data gathered from tissue specimen preparation represent the spatialarrangement of cells living within the boundaries of their mechanical,physiological and functional supportive extracellular matrix—thestructural formation of a given tissue. The architectural arrangement ofan organ's extracellular matrix serves, not only as boundaries betweencells and their tissue forming units, but is also a source of support atvarying levels of defined cellular organization.

Next, using Chroma Coded Manufacturing (CCM), chroma codes are createdto guide the production of the composite structures through the use ofcomputer aided design software to model the structure. CCM consists ofpoints, lines, and/or surfaces used in CAD to create three-dimensionalmodels of composite structures that are assigned colors, representativeof chromatically converted chroma codes embedded in the bio-CADblueprint to direct the necessary materials and conditions needed forthe manufacturing process. Chroma coding directs the accumulation andpositioning of factors needed for the manufacturing of and thestimulation of cellular constructs and their sub-cellular responses.Chroma coding enables models to provide detailed information guiding themanufacturing nests and paths for cells, during fabrication, creating astructural framework that both physically and biochemically guides thegenesis of the original organ structure. A three-dimensional tissueblueprint needs to be prepared from data on tissue development toorchestrate the release and selective attachment of functional peptidesto polymerize hydrogel events initiated by the computer integratedmicro-fabrication tools.

Chroma codes are created for compartments represented by a combinationof the numbers “0-9”. Within these compartments are sorted productiondata, such as the advanced materials being used in the manufacturingprocess; their three-dimensional placement by robotic positioning ofmaterials during production; the environmental conditions needed duringproduction for bio-manufacturing process; and the time intervals betweenprocesses within a manufacturing scheme. The resulting chroma codes arechromatically converted into colors, corresponding to the codes' seriesof ordered numbers. These numbers are embedded in the colors of thebio-CAD model and are retrieved from the design by a tri-stimuluscolorimeter and placed in the compartments assigned, in order toregulate the productions schemes in the robotically controlled devices.

The following is a list of steps to create a chroma code:

-   -   Chart the epigenetic information on the organ/tissue structure's        natural development/regeneration to correspond with the        available CAM tools, their specifications, construction        materials, and, where needed, the supported bioreactor        processes.    -   Script this chart into the bio-CAD code for a production plan        producing and transmitting signals that control the computer        aided manufacturing tools' robotic placement of construction        materials, robotic positions and triggering of laser tools for        molecular/atomic manipulation of materials, bio-support of        living fabricated structures using vascular and interstitial        fluid circulation.    -   Time the stimulation of tissue growth by regulating        environmental factors, forces applied to: temperature,        concentration of chemicals of developing structure using fluid        circulation, and laser motivated chemical reactions to        coordinate with a feedback sensor for temperature, pressure, and        chemical concentration of oxygen and carbon dioxide.    -   Map the expression locations and/or release locations of tissue        growth and/or differentiation factors and other chemical        engineering data needed to guide tissue production, within the        appropriate CAD structures.

Once the chroma codes have been completed and integrated into thebio-CAD, the bio-CAD will first code a vascular scaffold. This vascularscaffolding will be designed and made using three-dimensionalfabrication techniques, including the use of photon laser techniquesused in the micro-patterning of the molecular structures of materials.Computer-aided modeling of stereolithographic (STL) and micro-computertomographic (micro-CT) models enables the user to design the vasculartree system, including capillary beds. Reconstruction instereolithography format allows compatibility of micro-CT derivedthree-dimensional models with most CAD software. Noise from the datasetswill also be removed using interactive parallel visualization andgraphical analysis for contour plotting and generation of STL files. Bybeing able to reduce the noise, the memory footprint is also reduced,making the file smaller and more manageable. Additionally, corrosioncasting of luminal structures also provides accurate and high-resolutionresults for three-dimensional micro-CT reconstruction of capillary beds.Using a nanotip and nanopore array will influence cell differentiation,proliferation and growth. Using cylindrical circles of the nanotip andnanopore arrays will stimulate vascular tissue growth.

In other words, it begins by creating a bio-blueprint, also referred toas a bio-CAD, and identifying the tissue or organ to be transplanted orregenerated. The blueprint can then be designed, through reverseengineering, of the organ's connective tissue structure andextracellular matrix. This bio-informative data is used to producedevelopmental patterns which produce signals transmitted to the computeraided manufacturing or tissue engineering tools. The design willrepresent the extracellular matrix environment and stimulate thenecessary cell-type specific behaviors. Data instructs computer aidedmanufacturing tools, including bioreactors, spatially and temporally, toperform tasks during the fabrication process that allows the fabricatedstructure to experience environments designed to mimic molecularmechanisms and cellular interactions found in the natural development orregeneration of biological tissues.

Creating a functional three-dimensional tissue structure provides aplatform for the design and engineering to support the growth anddifferentiation of cells into their specific phenotypes to make up atissue or organ. The extracellular matrix is incorporated into thisplatform. The most important of the extracellular components that makeup the matrix, the basement membrane (or basal lamina), is the coresupport structure, and thus, the most critical component in theplatform. This design stimulates the necessary cell-type specificbehaviors. Tissue scaffolds can be designed using microscopic image datathat guide the generation of blastema-like cell masses and theirsubsequent regenerative processes. These scaffolds then guide andsupport the regeneration of the composite tissue structure.

The next step involves designing the three-dimensional models of theimage data, and incorporating molecular and environmental factors thatmediate cellular and subcellular behaviors, responsible for generatingthe tissue and/or organ structure. The process begins by coding a scriptcontaining bio-informative data and layering this information into abio-blueprint of the patterned response designed to mimic the chemicalengineering data gathered from a tissue's developmental process. Theblueprint sends scripted signals, via computer, to manufacturing toolsin progressive steps that are designed to create the regenerativeprocesses through the tools' responses. These responses are designed torespond to environmental feedbacks measured from fabricated structures.These activities are responsible for producing the changes in cellulardevelopment and regenerative processes. The fabricated structure willalso assist the molecular mechanisms which are the basis ofcommunication between cells themselves, and between cells and theirextracellular matrices, and are responsible for forming the germinalcell layers in humans: endoderm, ectoderm, and mesoderm and thesubsequent tissue types (for example: epithelium, skeletal muscle,cancellous bone) and their subsequent organ systems (for example:cardiovascular, endocrine, or nervous systems). These interactionsinclude molecules that interact, causing changes in molecularconfirmations which initiate G protein-coupled receptor mediated eventsthat culminate in the initiation of biochemical mechanisms on and withincell membranes, cytoplasm and the nucleus. By this means, the blueprintwill initiate and regulate the necessary cell processes with the cellorganelles that procure them into the tissue regenerative process.

Image data can also be obtained from reconstructing serial sectionsacquired from histological techniques, taken through a reference planecreated during tomography scans. Reference points are created in scannedtissue that corresponds with section tissues. The resulting images areimported into bio-CAD software where the afferent and efferentarterioles and venules mesh structures obtained with Micro-CT areconnected with mesh structures obtained through the reconstruction ofserial tissue sections from the capillary bed systems. Imagedata/information from tissue structures is used to createcomputer-assisted designs of these structures modeled to create astructural framework, both physically and biochemically for regeneratingthe original tissue structure around the vascular tree systems.Molecules (e.g. growth factors or cytokines) responsible for stimulatingoptimal cellular and sub-cellular responses are incorporated into thesevascular scaffolding designs.

The three-dimensional digital image is superimposed on athree-dimensional grid, having a plurality of axes in the X, Y, and Zdirections. There is no preferable distance between each axis. Thedistance should be determined based on the specification of the neededtissue or organ structure, and typically set to fall within theresolution of the robotics or materials needed in a particularfabrication scheme to produce a necessary structure in the scaffolddesign. Robotic printing is typically between five and two hundredmicrons, while laser fabrication or molecular manipulation can usedistances in a nanometer scale. There are variables to consider indetermining the distance between each axis including: the size of thematerial being robotically deposited, the spot size of the lasers beingused for photo initiated chemical activities such asphoto-polymerization of hydrogels into a network of tracts that guideand support cells in developmental precessions. Examples include, butare not limited to: blastema cells, mesenchymal cells,pericytes/fibroblast cells which migrate along the surface of scaffoldsdepositing fibronectin, collagen, and other molecules in theextracellular matrix. These distances are used to support thefabrication of the extracellular tracts and fine structures needed tosupport cellular behavior. The smaller the distance between axes, themore extracellular detail can be given in the scaffold, providing for anengrafted tissue with much finer detail.

Each place that is sectioned by the grid and having an intersectingpoint with the X, Y, and Z axes is given a designated point inthree-dimensional space, so as to provide a plurality of points. Eachpoint can then be identified as corresponding to a tissue type. Guidedblastemal formation is achieved by using coordinated spatial andtemporal molecular communication to dedifferentiate cells on surfaceinterfacing zones needing composite tissue regeneration. Guidedblastemal formation can also be attained by recruiting dedifferentiatedcells along three-dimensional paths and gradients that were prepared andinitiated by laser guided molecular manipulation and orchestrated bytissue engineering blueprints.

The bio-fabrication process involves signaling that is elicitedelectronically, chemically, and mechanically, and occurs betweencellular systems and technology. Computer aided manufacturing (CAM) usesthree-dimensional robotic bio-printers and multi-photon nanolasers.Bio-printers are used to strategically place cells and constructionmaterials from the bio-CAD blueprint's assigned locations. Multi-photonlasers are used to initiate chemical processes such as: thepolymerization of a hydrogel scaffold, the attachment of a chemicalcage/nanosphere, and/or the release of the contents encapsulated in saidspheres producing the desired developmental results.

The next steps include using the three-dimensional model to interfacecomputer programs for fabrication, and then using thosethree-dimensional models to direct micro-patterning and release ofmolecular factors that regulate cell migration, cell proliferation, celldifferentiation, and cell chemical production in three-dimensionalfabrications. Data/information is taken from tissue structures to createcomputer assisted designs of these structures. The computer aidedmanufacturing is used to fabricate a scaffold that is sectioned from thegrid discussed above. The program fabricates the scaffold by reproducingeach point from the three-dimensional image that is sectioned by thegrid as a corresponding point on the scaffold. Then a microcomputertomography image of vascular tissue is combined with the reconstructionof thin serial tissue sections. The resulting bio-CAD rendering producesa structural design for scaffolding on which a vascular tree can beconstructed.

Essentially, the scaffold is created by replicating the vascular treemodel in a solid form. Then the model is offset from its location withinthe septa to allow a model of the paralleling nerve fibers found in thetissue's septa. Next, the vascular tree model is replicated again andoffset in the septa to model the paralleling lymphatic vessels. Analternative to this step would be that the lymphatic system can be castand modeled uniquely. For example, tubular systems, such as kidneycollecting tubules, can be cast and modeled separately. The size andposition of the locations of each of the models to the locations modeledin the septa correspond to their natural size and location.

Next, use the three-dimensional model to direct three-dimensionalfabrication techniques to create structures that support and regulatetissue/organ genesis using the following procedure. Create structuralenvironments that regulate cell behavior, cell functions and patterntheir locations to correspond with image data/information taken fromtissue structures during the data collection and CAD modeling stages ofthis method. Create a structural framework using any three-dimensionalfabrication techniques. This includes but is not limited to the use ofphoton laser techniques used in the micro patterning of the molecularstructures of materials, such as hydrogels, and incorporating into thesestructures factors that are responsible for stimulating optimal cellularand sub-cellular responses that mediate cell behaviors.

Alterations should be made using computer directed two or three photonlasers, which alter the molecular structure of polymers in areas thatproduce the cellular responses such as migration or lack of cellpenetration, that outline the structural design formed in bio-CAD basedon the original image, molecular and environmental factors data acquiredduring the collection stages above. Use the three-dimensional model topattern specific molecular factors to form the capillary beds, which arevital to molecular exchange in and between cells living in and aroundvascular tree structures.

The three-dimensional model is then used to pattern the formation ofvascular trees, the capillary beds, extracellular matrix and cellsliving in and around them to form the basis for the three-dimensionaltissue growth necessary for organ development. Pattern cell migration,cell proliferation, cell differentiation and cell chemical production inthree-dimensional fabrications to correspond to the original image,including molecular and environmental factors, data acquired to createstructural environments that regulate cell behavior and cell functions.Use the image data/information taken from tissue structures to createcomputer assisted designs of these structures modeled to create astructural framework, both physically and biochemically for tissuegenesis of the original structure.

In building the various scaffolds, bio-blueprints are used to guide thecreation of structural and chemical environments that regulate cellularbehaviors and/or functions. Many combinations of steps will result inthe formation and/or production of composite tissues based onthree-dimensions. Some examples of the interrelationships between stepsand this method include but are not limited to the followingrelationships:

-   -   Directing computer programs that alter the molecular structure        of polymers in areas that produce the cellular responses based        on the three-dimensional model;    -   Directing the housing and releasing of factors that are        responsible for the cell's morphogenesis, based on a        three-dimensional model;    -   Directing computer programs to direct factors to control cell        production and/or the release of growth factors, cytokines, and        chemokines based on a three-dimensional model;    -   Directing the regulation of migration, proliferation,        differentiation, and chemical production of cells based on a        three-dimensional model;    -   Directing organ genesis in a vascular bed chamber, based on a        three-dimensional model;    -   Directing fabrication of the chosen organ tissues in body        cavities, in wounds, and surgically created spaces based on a        three-dimensional model.

The next step uses the three-dimensional models to direct compositetissue regeneration. The vascular bed chamber regulates vascular flowand media content using sensors that generate and feed data back to acomputer capable of regulating the dispensing of molecular andenvironmental factors that control cell behaviors such as migration,proliferation, differentiation and chemical production in a bio-reactiveenvironment.

CAD models of casts representing a vascular tree's lumen are used toprovide the framework for data concerning sizes and dimensions that willdirect fabrication of scaffolds in a vascular bed chamber capable ofestablishing tissues that mimic the original vascular tree. In thevascular bed chamber, a volume flow pumped to simulate a vascularcirculatory system creates variable forces on the tissue structure.Combined with external structural supportive forces, the vascular bedchamber regulates the structural properties of developing blood vesselsand surrounding tissues.

Following this, the three-dimensional model is used to directfabrication of the chosen tissue/organ in body cavities, wounds,surgically created spaces, or directly in a vascular bed chamber. First,alter the structure of reactive polymers such as photosensitivepolymers, which are placed into a body cavity, and/or wound, and/or asurgically created space, and/or vascular bed chamber—in patternsconsistent with the three-dimensional models.

Then, use injectable polymers as a foundation for structuralenvironments that regulate cell behavior, cell functions, and patterntheir locations to correspond with image data/information taken fromtissue structures during the data collection and CAD modeling stages ofthis method. Polymers are altered in three-dimensional patterns,directed by the bio-CAD model to create a structural framework using anythree-dimensional fabrication technique. This includes but is notlimited to the use of photon laser techniques used in the micropatterning of the molecular structures of materials, such as hydrogels,and incorporates into these structures factors that are responsible forstimulating optimal cellular and sub-cellular responses that mediatecell behaviors.

Make alterations using computer directed two or three photon lasers,which alter the molecular structure of polymers in areas that producethe cellular responses, such as migration or lack of cell penetration,which outline factors of molecular and environmental data acquiredduring the collection stages above, and regulates in patterns consistentwith three-dimensional models initiating the formation of naturalvascular tree structure and other tissue structures, such as alveolarsacks or nerve tracts, in body cavities, in wounds or in surgicallycreated spaces.

Use the three-dimensional model to pattern specific molecular factors toform the capillary beds, which are vital to molecular exchange in andbetween cells, living in and around vascular tree structures in bodycavities, in wounds, in surgically created spaces or in one of thevascular bed chamber configurations. Then, use the three-dimensionalmodel to pattern the formation of vascular trees, the capillary beds,extracellular matrix and cells living in and around them to form thebasis for the three-dimensional tissue growth necessary for organdevelopment in body cavities, in wounds, in surgically created spaces,or in a vascular bed chamber. Pattern cell migration, cellproliferation, cell differentiation and cell chemical production tocorrespond to the original image, including molecular and environmentalfactors data acquired to create structural environments that regulatecell behavior and cell functions in body cavities, in wounds, insurgically created spaces or in a vascular bed chamber. Usethree-dimensional model to direct the fabrication of physical andbiochemical structural frameworks that stimulate regeneration of organstructure in body cavities, in wounds, in surgically created spaces, orin a vascular bed chamber.

Next, use the three-dimensional modeling computer program to direct thealteration of the molecular structure of polymers in areas that producecellular responses. Alter the structure of polymers in three-dimensionalpatterns, directed by the bio-CAD model, using image data/informationtaken from computer assisted designs of the needed tissue structures.Design a model to replicate the structural framework, both physicallyand biochemically, for tissue genesis of the original structure. Then,build around vascular tree systems formed in accordance with thepreviously described process of vascular scaffold designing. Fabricate,directly from three-dimensional image data, an in vivo occurringvascular network that includes the capillary systems, which arenecessary to feed the surrounding tissues. Incorporate into thesedesigns using any three-dimensional laser, two or three photon lasertechniques, factors which are responsible for stimulating optimalcellular and sub-cellular responses that mediate both normal andabnormal cell behaviors that outline the natural vascular tree structureor other tissue structures such as alveolar sacks or nerve tracts.

Next, using the three-dimensional model, house and release previouslydescribed molecular factors for cell morphogenesis in fabricated photoreactive material. Using the three-dimensional model, trigger therelease using laser patterning of factors responsible for cellularmorphogenesis based on spatial and temporal patterns of growth factorsignaling in order to form tissue structures such as the tunic layersfound in blood vessel walls.

The chroma coded bio-CAD will plot the position, the photopolymerization, and photo activated release of molecularly cageddevelopmental factors, by robotically positioning the photon emittersusing multi-photon femtosecond lasers and robotic dispensing tools. Itis best to use a femto tunable laser based STED system in combinationwith a scanning confocal microscope. STED technology is used to overcomeany diffraction limitations for multifunctional instruments.

STED is an implementation of a family of scanning far-field opticaltechniques and extends a confocal laser scanning microscope by providingan additional illumination path for fluorescence depletion. STED usestwo laser beams having different wavelengths (or colors), instead of asingle source of light in conventional photolithography. Typically, twolasers are used, violet—the normal excitation laser, and green—having adonut configuration. The green laser depletes all of the fluorescencegenerated by the excitation light and only the tiny middle hole willradiate fluorescence. This unique system can overcome conventionaldiffraction limits to achieve nanoscale spatial resolution for thefabrication of nanostructures.

When the activation beam has a higher laser power and activates theresist to cause cross-link to make patterns on the resist layer, theinhibition beam suppresses the activation while maintaining a valley atthe center of the activating beam. The outcome of the activation peakand “deactivation” donut pattern gives a spatial dosing pattern that issubstantially smaller than the far-field diffraction pattern of atightly focused optical beam. The deactivation of the resist isreversible, i.e. when the deactivation light is removed, the resist canagain photo-polymerize when it is exposed to the excitation light. Thus,nanostructure can be patterned by scanning the laser beams on theresist. The activation and deactivation can be achieved by optimizingthe laser intensity and timing of the laser pulse. The deactivation beamcontrols the spatial distribution of exposure, and thus the resolution.Furthermore, the donut inner diameter can be decreased by increasing theintensity of the donut beam. In this way the resolution of patterningcan be significantly increased to overcome diffraction limit and patternnanostructures.

The method discussed above can be used to generate tissues bothexternally, for transplantation, or directly on/in the human body, forregeneration. This section will focus on the intricacies associated withthe method in relationship to the regenerative process and using thesecond configuration of the vascular bed chamber.

The bio-CAD is formed by converting three-dimensional CT scans of thewound or defect, in order to produce custom scaffolds to fit thespecific defects. The blueprint is designed to produce athree-dimensional substrate which will guide the cells in the body toform a regenerative blastema for the organ or tissue to be regenerated.The subsequent blastema is guided and sustained in a form fittedregenerative chamber, the second embodiment of the vascular bed chamber(VBC-II) described below, where it generates the necessary composite oftissues needed to replace missing, damaged, or diseased organs ortissues.

The regenerative process is promoted by creating three-dimensionalstromal and cellular structures along with the appropriate embeddedsignaling or trophic molecules to mimic the blastema-associatedregeneration process. The initial signals for regeneration are growthfactors. The initial target of nerve signaling is the apical woundepithelium This epithelium is functionally equivalent to the apicalepithelial cap in developing limbs, and is required for both outgrowthand pattern formation. Apical epithelial cap function is dependent onbasal keratinocytes withdrawing from the cell cycle, and that cell cyclewithdraw is induced by nerve signaling and is maintained by gapjunctions between adjacent basal keratinocytes. Expression of the markerfor apical epithelial cap dedifferentiation can be induced bykeratinocyte growth factor (or FGF7), which is produced by injured orregenerating nerves. The downstream targets of nerve or apicalepithelial cap signaling are the mesenchymal cells of the blastema. Thegeneration of a blastema at the site of injury is a critical step forlimb replacement. A blastemal-inducing film (discussed below) is createdand placed over the wound. The bio-CAD blueprint will aid to signal andregulate the growth factors in the proper time and space, promotingregeneration directly on or in a human body.

A synthetic blastema is stimulated to form along the margins of theVBC-II (the construction of which is discussed below) and ties into themain vascular supply for the region to be regenerated. The VBC-II isplaced on the wound or in the body and attached to bypassed bloodvessels and acted upon by the bio-CAD blueprint. This enactmentestablishes the complex biological interrelationships between cellsresponsible for the formation of tissues.

The VBC-II is robotically filled with photo-polymerizable hydrogel, orany other material capable of acting as a supportive scaffold. TheVBC-II is also filled with encapsulated dedifferentiation,differentiation, and growth factors to be attached as peptides, bylasers, to the polymerized hydrogel or released freely as chemotaxisfactors, attracting pluripotent adult stem cells into the degeneratingcontainer of polymerized hydrogel by both holographic and multiphotonlasers robotically controlled by the bio-CAD blueprint. Once completed,a porous formfitting cover is applied to help, where necessary, with theincubation process of tissues including, but not limited to: skin andother exposed tissues or organs when needed for healing process.

Making the Production Chamber

A production or inversion chamber 100 may be used to aid in the functionof the first configuration of the vascular bed chamber (VBC-I) 10, asshown in FIG. 5. The production chamber 100 may be any shape or size aslong as it encompasses the entirety of the VBC-I 10. Ideally, theproduction chamber 100 will be square or rectangular in shape and madefrom a transparent material, so that the user may easily see theproduction as it progresses. The base of the production chamber musthave a square, transparent section in the middle 130, so that the lasersand microscopes may pass through to the production area of the VBC-I 10.The outer portion of the base 122 may be thermal regulated and a tightseal 132 made between the outer thermal regulated portion 122 of thebase and the transparent middle section 130 of the base. The chamber 100is designed so that VBC-I 10 is placed upside down within the chamber100. The widest portion, the top of VBC-I 12, will be at the bottom 122and the telescoping sides 14-20 will be raised in an inverse conicalshape, as the base of VBC-I 22 is raised by clamps 108 attached to thesupport rod 102 in the production chamber 100. The chamber 100 may havespring loaded clamps 110 on the base 122 that allow the lip of the VBC-I10 to attach and be stabilized to the base 122 of the production chamber100.

Additionally, the production chamber 100 has a support rod 102 that runsthe length of the chamber and is adjustable in the vertical plane. Therod 102 is attached to an adjustable slider 112 on the sides of theproduction chamber 100. The adjustable slider 112 is allowed to slidebecause of a sleeve support 104 on the side of the chamber 100, and amotorized knob 120 allowing the chamber 100 to be completely enclosed atall times. Attached to the rod 102 are clamps 108 that are adjustablealong the length of the rod 102, to accommodate the various sizes inwhich the VBC-I 10 can be made. A clear illustration of the relationshipbetween the upper clamps 108 and their attachment to the support rod 102by way of an adjustable slider 106 is shown in FIG. 4. The upper clamps108 are supported by an adjustable slider 106 attached to the supportrod 102 to allow the clamps 108 to slide in the horizontal plane, thusaccommodating any base 22 size of VBC-I 10. The rod 102 is attached to asleeve 104 that allows it to move up and down within the chamber 100;however, any method of raising or lowering can be used. The rod 102 islowered down to the inverted base 22 of the VBC-I 10, and the clamps 108along the rod 102 can be held in place by adjustable/motorized knobs120. During production, a motorized knob 120 allows the rod 102 to movevertically up, thus raising the telescoping sides 14-20 of the VBC-I 10to raise as the tissue or organ grows larger so that the tissue or organis completely enclosed in the VBC-I 10.

The production chamber 100 should have at least as many input ports 114as the corresponding VBC-I 10 being used with it. Ideally, theproduction chamber will have at least three input/output ports 114, 116,124, 126, 128, where 128 is an example of an optional placement oradditional placement port. These ports 114, 116, 124, 126, 128 allow forthe exchange and flow of materials into and out of the chamber,continuing into the VBC-I 10, during and after production to aid inkeeping the tissue or organ viable for transplantation or research.These ports 114, 116, 124, 126, 128 are designed to connect into theVBC-I 10. while it is in an inverted state.

Making VBC-I

The Vascular Bed Chamber-I (VBC-I) 10 is a receptacle that istelescopically increased in its height, robotically, to match thethree-dimensional bio-fabrication process of tissue structures. This isdone while simultaneously providing for the exchange of liquid mediumsand materials within the fabricated structure and in the areassurrounding the tissue structure's production. It does this whileproviding for the tissue's support and maturation during thebio-fabrication process. It can also assist in regulating the buoyanceof structural materials added to the VBC-I 10 by allowing changing offluids of variable viscosities, supplied to the production area byperipheral devices, computer regulated and prearranged by the tissue'sBio-blueprint.

The VCB-I 10 device is made from cost effective materials and can memade of anything from biodegradable glucose polymers, protein polymers,glass or plastics, to stainless steel. Its parts can be producedseparately by traditional casting, machining, drilling, molding andshaping means and assembled as described below, or three-dimensionallyfabricated from a CAD model.

Its structure may be transparent, to allow during the tissue structuresmaturation phase, the laser manipulation of chemicals, materials andcells, robotically placed within the chamber during the structure'sinitial fabrication processes, or being supplied to the bio-fabricationsite during tissues maturation phase through the VBC-I's entry ports 24a, 26 a, 28 b; or may be opaque to prefer light from affect thebio-fabrication of the maturing tissue structures.

FIG. 1 shows a cross section of VBC-I 10, upright and in its expandedstate. This figure shows the interior and how the inflow and outflowtubes 24, 26, 28 are constructed and function within the VBC-I 10. Thewalls of the VBC-I 10 are constructed of tapered sections 14-20 thatmove along each other telescopically in height as the top section islifted during the initial production phase. The diameter of distanceacross the top pf each section, ideally, is 2 mm larger than the sectionfollowing below its position. The diameter of distance across the bottomof each section, ideally, is 4 mm smaller than its top opening. Exceptfor the top and bottom sections, the wall of each section, ideally, is 9mm in height and tapered to meet the 4 mm difference in diameter betweenits upper and lower openings.

The very top section, ideally, is 3 mm taller than the middle sectionsto provide for protruding lipped structure 12 used to catch hold ofclamps 110 that hold it in place for its vertical lifting. Two clamps108 for lifting are constructed with inner facing surfaces shaped to thecontour of the wall of the VBC-I's base 22.

The top section lip 12 also provides for the snug fit of a cap for VBC-I10, which can be applied when the initial fabrications stages arecomplete at the tissue's maturation stage, when it required. The capaffixed to the top section 12 has an entry port 26 a in its top for atube that connects to the bio-fabricating tissue's main input/outputvessel, as illustrated in FIG. 2. The tube 26 a may have adjustablesliding clamps/locking spacers 38 on both sides of the cap that allowmodifiable depths of the tube's descent into the VBC-I 10.

The very top section also has an input port 24 a with an outflow tube 28that enters at an angle parallel to the VBC-I's base 22. The tubes' 24a, 28 openings are manufactured to fit flush with this section's innerwall, allowing its smooth telescopic movement across the outer wall ofthe lower connecting section. This port 28 allows for bottom upextraction/out flow of contents surrounding the bio-fabricating tissuestructures.

The wall of the bottom section 20 is cupped with an inner depth of 7 mm,ideally, and an outer height of 9 mm, ideally. The bottom section 20 isattached to an appropriate base 22 at the time of the VBC-I's 10assembly. The attachment of the base 22, done after all of the taperedwalled sections 14-20 are placed in the order of their width, withineach other, hold all the sections in place. In order to achieve this,the base 22 must at least be 4 mm wider that the lower opening of thetop section 12. The bottom of the base 22 is shaped to match thespecimen mounting mechanism centrally located on the robotic productionstage.

Ideally, the base 22 may have a wall with an inner depth of 7 mm. Thebase may have two inlet tubes 24 b, 26 b that enter the VBC-I 10perpendicular from the bottom and one outlet tube 28 a that enters at aright angle from the side. The side outlet tube transverses the wall ofVBC-I's base 22 and enters the wall of the VBC-I 10 within a fewmillimeters of its bottom 22. The side tube protrudes slightly into 28 bVBC-I's 10 open area, allowing a regulated, top down extraction/out flowof contents surrounding the bio-fabricating tissue structures.

FIG. 6 illustrates the VBC-I 10 in an upside down, expanded state—thearrangement it would be in after production initially finishes. The twobottom ports 24 b, 26 b have attached tubes which enter at a right angleto the floor 22 of the VBC-I 10. These tubes 24 b, 26 b may be curvedfor convenience as they extend away from their entry points, allowingtheir clearance of the robotic production stage from a suspendedposition in its specimen mounting mechanism. One tube 26 is centrallylocated and protrudes about 5 mm, ideally, into the open area of theVBC-I 10. This tube is used for the variable supply of production,maintenance and regulatory fluids, materials and cells, to thefabrication sites (interior end of 26 b) within the tissues by way of amain inlet blood vessel, fabricated from materials attached to tube'souter wall interior end of 26 b during the initial bio-fabrication ofthe desired tissue structures. The second tube 28 meets flush with thebottom floor of the VBC-I 10. Its entry point is off to the sideallowing a variable and independent flow of fluids and materialdiscussed above, for the purposes disused above, form outward positionsinto the bio-fabrication sites.

Making VBC-2

A second configuration of the vascular bed chamber (hereinafterreferenced as VBC-II) 200 is intended for tissue, organ, or completelimb regeneration directly on a patient's body/wound 202. FIG. 7illustrates a complete setup of the VBC-II in a state ready forproduction of a new tissue or organ. The VBC-II 200 is a fitted chamber,produced specifically to fit the patient and the tissue/organ/limb 202to be regenerated. The chamber 200 serves as an interface with thepatient's body's regenerative activities, the bio-stimulatory scaffolddescribed above, and the ex vivo robotically controlled systems. Thechamber 200 is needed to aid in the stimulation and support of theregenerative processes designed in the method. The chamber 200coordinates the interface of computer aided manufacturing,bio-stimulatory and tissue regeneration through the chroma code,incorporated into the selected blueprint.

The chamber of the VBC-II 200 is designed for construction in individualsegments 206 that may be transparent in nature. Segmenting the chamberallows the user to customize the size and shape to fit thebiofabrication site and tissue structure requirements for patientspecific needs. The segments interlock along the sides 238, 240,alternating male and female connectors, as shown in FIG. 10. FIGS. 12and 13 illustrate the underside of each segment being lined with inflowholes 242 that lead to channels 236 within the segments 206 to allow forrecirculation and extraction of fluids and materials that aid inregeneration. At the end of each segment 206 are outflow ports 218 toaid in the recirculation of fluids and materials through the chamber.These ports 218 should have a threaded end 216 to allow for theattachment of additional segments 206 or accessory attachments, such asa small chamber for the regeneration of digits 244. FIG. 14 illustratesthis digit attachment 244 having one inflow port 242 that attaches to asingle segment 206 by way of the interlocking, preferably screw, ends216. Inside of the digit attachment chamber are holes 218 that allow forthe outflow and recirculation of fluids and other biological materials.

To use VBC-II 200 with the method described above, the user must createa blastemal-inducing film 204. This can be created using a waxy paper224, laying down a fatty acid mixture containing intergins 222 andoverlaying the mixture with a protective hydrophilic gauze 220, as shownin FIG. 8. This creates two regions, a hydrophilic region 228 and ahydrophobic region 230 separated into their own compartments 226. Thisfilm 204 must be molded to fit over the patient's wound 202 area inorder to begin the regenerative process. The best way is to heat thefilm 204 over a mold 232 having been created to fit the patient's wound202, the mold illustrated in FIG. 9. FIG. 9 illustrates the mold 232 ofthe wound 202 with the blastemal film 204 overlaying it. The mold 232 isthen heated, preferably from an internal heating component 234, but maybe done with any heating method. The heat allows the film 204 to fit themold 232 of the wound 202 so that it will fit securely over the wound202 directly on the patient's body.

Once the film 204 has been created and inserted on or inside of thepatient's body in the area to be regenerated, the segments 206 can beattached together and overlay the area to be regenerated. Next, a tubeis inserted perpendicular to the film. This tube will be the fabricationsite, where the cells are placed to regenerate the affected area on thepatient. FIG. 14 shows the tube comprised of several sections. At thevery end of the tube, where it meets the film 204, are tubules 246 thatlay over the blastema film 204. The tube may expand telescopically, onesection 212 being slightly larger than the other 208, allowing the tubeto collapse down for easy and damage-free removal. FIG. 15 illustratesthe tube in its collapsed state. The tubules 246 may be drawn up thesides of the small half of the tube 208, the bottom half. Then the tophalf of the tube 212, preferably having a lip 214, will slide over thebottom half 208 and the tubules 246, completely encasing them so that asingular tube is removed from the site once regeneration is complete.The tube allows for the inflow, through the top section 212 down to thebottom section 208 and finally through the tubules 246, of materials anddisperses the materials through the outflow ports 248 located on eachtubule 246.

Finally, the laser 210 that will aid in production will be placed.Ideally, it is used at a 45-degree angle to the inflow tube that servesas the fabrication site, as illustrated in FIG. 7. FIGS. 16 and 17illustrate the laser fabrication goggle 254 that will be fitted over thelaser 210. This is a rounded chamber designed to fit over the microscopeobjective lens end of the laser 210. The goggle 254 should have femalethreads at the top 250 to allow it to screw onto the laser.Additionally, the bottom of the chamber should have at least atransparent section in the middle 252 that will allow the light from thelaser to pass through. FIG. 17 illustrates the entire assembly, with thelaser 210 in the goggle 254, having been screwed into the top 250, withthe laser sitting in immersion oil. Once the laser 210 has been placed,VBC-II 200 is considered to be completely assembled, andfabrication/regeneration can begin.

What is claimed is:
 1. A method of creating and keeping artificialtissue alive, comprising: selecting a tissue for production, isolating avascular system of said tissue using a vascular casting, isolating asepta system of said tissue, capturing an image of said vascular andsepta systems, importing a three-dimensional image, obtained from saidimages into a computer-aided design software program, repairing saidimage using computer-aided design (CAD) tools and converting mesh intonon-uniform rational B-spline models, designating the boundary where thesepta and vascular cast interface cells as site of a basement membranesystem, using computer-aided design tools to clean up said basementmembrane system and modeling the walls of the vascular system for aproduction scheme, corresponding the models within the septa system ofsaid tissue to said production, creating a Chromatic Representation OfMultiple Alignments (CHROMA) code to provide for factors required toproduce tissue structures, mapping the expression of growth factors,creating a bio-CAD blueprint by: superimposing the three-dimensionalimage on a three-dimensional grid having a plurality of axis x, y, and zdirection, wherein each point of intersection between the x, y and zaxes is an x, y, and z coordinate, assigning each point of intersectionat least one tissue type, converting the three-dimensional image into ascaffold for an engrafted tissue, wherein the scaffold has a pluralityof regions suitable for supporting cell growth, using thethree-dimensional image to form patterns of the vascular system andextracellular matrix as the basis for the three-dimensional tissuegrowth during organ development; and patterning cell migration, cellproliferation, cell differentiation, cell chemical production for athree-dimensional manufacturing process by corresponding to the originalthree-dimensional image to regulate cell behavior and function,modifying bio-CAD blueprint to create paths and locations for thethree-dimensional manufacturing process, duplicating the bio-CASblueprint, using the bio-CAD blueprint to apply coordinates to eachsub-model for conveying scripted roles to computer-aided manufacturinginstrument, associating the production scheme to appropriate saidcoordinates applied to each sub-model using programs that guidecomputer-automated test engineering and computer-aided manufacturingtools by: coding the production scheme for each uniquely modeled tissuestructure by creating a numbered table; assigning the code to materialsand processes used in the production scheme for the three-dimensionalmanufacturing process; dividing the table into six categories, eachrepresented by an equal number of digits; assigning categories to eachof the following color components: hue, saturation, value, red, green,and blue; using common graphic software color picking tools to convertthe code into color; applying the computer color to the boundaryrepresentation of each set of coordinates; using a computer to takeinstruction from the bio-CAD blueprint, by decoding the color values atthe assigned coordinates, to coordinate the delivery of conditionedgrowth media, cells, chemotactic and developmental growth factors in arobotically controlled manufacturing process, and print an authentic,living tissue in the computer-aided manufacturing instrument.
 2. Themethod of claim 1, further comprising, Injecting reactive polymers intothe body cavities, wounds, and surgically created spaces, Altering themolecular structure of the injected polymers to initiate formation of avascular system using the three-dimensional-image, and Creating livingtissue inside or on a human body.
 3. The method claim 1 furthercomprising Printing living tissue inside a chamber with flows of fluids,cells, and growth factors, said chamber comprising at least one inflowand outflow port that will allow fluids and other biological materialsto circulate, wherein said chamber is constructed from biodegradablepolymers, glass, plastics, and/or metals, and is configured in any shapethat will house the tissue or to be created and with space for flowingfluids and biological materials through the chamber.